Isooctyl palmitate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isooctyl palmitate

EC Number:

215-675-9

EC Name:

Isooctyl palmitate

Cas Number:

1341-38-4

Molecular formula:

C24H48O2

IUPAC Name:

6-methylheptyl hexadecanoate

Test material form:

liquid

Remarks:

a colourless liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

A range-finding test was conducted to find the concentrations that cause algae growth inhibition. Based on the results of the range-finding test, a definitive test was carried out with the following concentrations of test Item : 0 (control); 1; 3,2; 10; 32 and 100 mg.L-1.

Test solutions

Vehicle:

no

Details on test solutions:

The stock solution used in the range-finding test was prepared by dissolving 0.0250 g of the test item in 250 mL of with OECD medium, resulting in a concentration of 100 mg.L-1. To assist solubilization, the solution was stirred for 20 minutes in a magnetic stirrer and 20 minutes of ultrasonic dispersion was also applied. The stock solution of 0.1; 1; 10 and 100 mg.L-1 were prepared from dilution of the stock solution.

The stock solution used in the definitive test was prepared by dissolving 0.1000 g of the test item in 1000 mL of with OECD medium, resulting in a concentration of 100 mg.L-1. To assist solubilization, the solution was stirred for 20 minutes in a magnetic stirrer and 20 minutes of ultrasonic dispersion was also applied. The stock solution of 1; 3.2; 10; 32 and 100 mg.L-1 were prepared from dilution of the stock solution.

From these initial stock solutions, a series of dilutions was prepared in order to obtain the desired tested concentrations (Tables 3 and 4).

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test system was obtained from Culture Collection of Algae at Goettingen University - SAG 61.81 on October, 08th, 2013, and is recommended by the Guideline OECD 201 (2011).
The stock culture was prepared and maintained under lab-controlled conditions in solid medium with protease peptone. Its composition is described in Table 1.

The inoculated solid medium was kept inside a culture room, with controlled temperature of 21 to 24°C and luminosity of 2100  10% lux. After algae cultures reached an ideal growth (about one week), they were maintained at temperature of 2 to 6°C and luminosity of 100 to 200 lux in order to slow down the algae growth.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.0 to 22.2 ℃

Details on test conditions:

A range-finding test was carried out using the following concentrations of test item: control; 0.1; 1; 10 and 10 mg.L-1 of test Item, with two replicates per concentration. The algae inoculum was transferred aseptically to each test flask, with calculated volume to yield an initial cell concentration of 5 x 103 cells.mL-1.

After 72 hours, samples of each test flask were taken in order to count the cells and to determine the concentrations, using a Neubauer chamber and a microscope, that caused algal growth reduction.

Based on the results obtained in the range-finding test, the definitive test was carried out with the following concentrations of test item: control; 1; 3.2; 10; 32 and 100 mg.L-1. The test was performed with six replicates per each concentration and control, therefore tree replicate was prepared for determinations of pH and chemical analysis. The algae inoculum was transferred aseptically to each test flask, with calculated volume to yield an initial cell concentration of 5 x 103 cells.mL-1.

All test flasks were randomly positioned in an incubating chamber with controlled temperature and kept under continuous shaking and lighting conditions. Every day the flasks were relocated to different places inside the incubating chamber.

Tables 3 and 4 show the stock solution and medium volumes used to obtain the concentrations of the range-finding and definitive tests.

The definitive test was carried out under the conditions described below:

Temperature: 22.0 to 22.2oC
Light: 6.126 lux (average)
Photoperiod: continuous light
Shaker velocity: 130.6 rpm (average)
Test duration: 72 hours

Temperature was measured daily using a digital thermometer. The pH was measured at the beginning of the test and at 72 hours (Table 8).

One aliquot was taken from each test flask in order to determine the algal growth over the period of 24, 48 and 72 hours after the start of the test. A Neubauer chamber and a microscope were used to determine the cell number (cells.mL-1).

A test with the reference item, potassium dichromate, was also carried out in order to verify the sensitivity of the test system. The procedure for this test was the same as for the definitive test described above, with initial cell concentration of 5 x 103 cells.mL-1 for each concentration and using nominal concentrations of 0 (control); 0.18; 0.32; 0.56; 1.00 and 1.80 mg.L-1 and two replicates for each test concentrations.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.2 mg/L

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 23.32 mg/L

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1 mg/L

Basis for effect:

growth rate

Details on results:

The 72-hours EyC50 value of test item, obtained in this study with Pseudokirchneriella subcapitata, was 23.32 mg.L-1 (95% confidence limits = 17.06 to 31.87 mg.L-1) and the 72-hours ErC50 value was not determined because the highest tested concentration (100 mg.L-1) caused only 22% of the inhibition in the algae growth, therefore, the 72-hours EyC50 value was greater than 100 mg.L-1. The highest concentration of the test item in which was not observed any statistically significant adverse effect on algal growth (NOEC) over the test period (72 hours) was 3.2 mg.L-1 and the lowest test item concentration at which was observed significant reducing effect on growth (LOEC) was 1 mg.L-1.

Results with reference substance (positive control):

The 72-hours EyC50 value of the reference item potassium dichromate was 0.66 mg.L-1 (confidence limits at 95% = 0.60 to 0.72 mg.L-1), demonstrating that the toxic sensitivity of the algae was within the accepted range (ISO, 1989), which report toxicity ranged between 0.60 to 1.03 mg.L-1.

Any other information on results incl. tables

In order to check the validity of the test, the biomass in the control cultures should increase exponentially by a factor of at least 16 within 72 hours. The mean coefficient of variation (CV) for section-by-section specific growth rates in the control cultures must not exceed 35% and the CV of average specific growth rate during the entire test period in replicate control cultures must not exceed 7%. In fact, in the present study, the biomass in the control cultures increased 1119 times at end of the test (Table 9). The mean coefficient of variation (CV) of the growth ratein the control group was 28.7% (Table 10) and the CV of average specific growth rate during the entire test period in replicate control cultures was 0.2% (Table 11), showing therefore, the validity of this study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72-hours EyC50 value was 23,32 mg.L-1 and the 72-hours ErC50 value was not determined because the highest tested concentration (100 mg.L-1) caused only 22% of the inhibition in the algae growth, therefore, the 72-hours EyC50 value was greater than 100 mg.L-1. The highest test Item concentration which no observed significant adverse effect (NOEC) on algal growth was 3.2 mg.L-1 and the lowest test Item concentration at which was observed significant reducing effect on growth (LOEC) was 1 mg.L-1.